Novel melanocortin-4 receptor sequences and screening assays to identify compounds useful in regulating animal appetite and metabolic rate

Abstract

The present invention relates to novel nucleic acids encoding canine and feline melanocortin 4 receptors and their gene products. Furthermore, the present invention relates to screening assays to identify compounds that modulate the activity or expression of the melanocortin 4 receptors of the invention. In addition, the present invention relates to methods and therapeutic compositions for the treatment of appetite-related, metabolic and reproductive disorders related to inadequate food intake and energy metabolism, comprising administering to animals compounds that modulate the activity or expression of melanocortin receptors. In one aspect, the invention relates to methods and compositions that antagonize the activity or expression of melanocortin 4 receptors in order to enhance the appetite of diseased, stressed or injured companion animals, livestock or poultry comprising administering compounds that antagonize the activity or expression of the novel melanocortin 4 receptors of the present invention. In another aspect, the invention relates to methods and compositions that agonize the activity or expression of melanocortin 4 receptors in order to treat, e.g., obesity of companion animals, such as cats and dogs comprising administering compounds that agonize the activity or expression of the novel melanocortin 4 receptors of the present invention.

1 . An isolated nucleic acid encoding a functional MC4R, or the complement thereof, said nucleic acid comprising a nucleotide sequence selected from the group consisting of: 
 (a) a nucleotide sequence which hybridizes under conditions of moderate stringency to the coding region of SEQ ID NO:1;    (b) a nucleotide sequence which hybridizes under conditions of moderate stringency to a polynucleotide which is complementary to the coding region of SEQ ID NO:1;    (c) a nucleotide sequence which hybridizes under conditions of moderate stringency to the coding region of the feline MC4R as deposited with the ATCC and having ATCC Accession No. PTA-1762; and    (d) a nucleotide sequence which hybridizes under conditions of moderate stringency to a polynucleotide which is complementary to the coding region of the feline MC4R as deposited with the ATCC and having ATCC Accession No. PTA-1762,    with the proviso that said functional MC4R is not human, porcine, murine, rat or chicken.    
     
     
         2 . An isolated nucleic acid encoding a functional MC4R, or the complement thereof, said nucleic acid comprising a nucleotide sequence selected from the group consisting of: 
 (a) a nucleotide sequence which hybridizes under conditions of high stringency to the coding region of SEQ ID NO:1;    (b) a nucleotide sequence which hybridizes under conditions of high stringency to a polynucleotide which is complementary to the coding region of SEQ ID NO:1;    (c) a nucleotide sequence which hybridizes under conditions of high stringency to the coding region of the feline MC4R as deposited with the ATCC and having ATCC Accession No. PTA-1762; and    (d) a nucleotide sequence which hybridizes under conditions of high stringency to a polynucleotide which is complementary to the coding region of the feline MC4R as deposited with the ATCC and having ATCC Accession No. PTA-1762.    
     
     
         3 . An isolated nucleic acid comprising a nucleotide sequence that: 
 (a) encodes a polypeptide according to SEQ ID NO:3; or    (b) encodes a polypeptide encoded by the feline MC4R clone as deposited with the ATCC and having ATCC Accession No. PTA-1762.    
     
     
         4 . The isolated nucleic acid of  claim 3 , wherein said nucleic acid has a nucleotide sequence according to SEQ ID NO:1 or the feline MC4R clone as deposited with the ATCC and having ATCC Accession No. PTA-1762.  
     
     
         5 . An isolated nucleic acid comprising a nucleotide sequence having more than 87.4% identity to SEQ ID NO:1 or the feline MC4R clone as deposited with the ATCC and having ATCC Accession No. PTA-1762.  
     
     
         6 . An isolated nucleic acid comprising a nucleotide sequence encoding a polypeptide having more than 98.2% identity to SEQ ID NO:3 or the polypeptide encoded by the feline MC4R clone as deposited with the ATCC and having ATCC Accession No. PTA-1762.  
     
     
         7 . An isolated nucleic acid comprising a nucleotide sequence encoding an ECD of a feline MC4R corresponding to amino acids 1-46, 96-123, 186-190, or 268-279 of SEQ ID NO:3 or of the polypeptide encoded by the feline MC4R clone as deposited with the ATCC and having ATCC Accession No. PTA-1762.  
     
     
         8 . An isolated nucleic acid comprising a nucleotide sequence encoding a CD of a feline MC4R corresponding to amino acids 70-76, 146-165, 212-244, or 302-333 of SEQ ID NO:3 or of the polypeptide encoded by the feline MC4R clone as deposited with the ATCC and having ATCC Accession No. PTA-1762.  
     
     
         9 . An isolated nucleic acid encoding a functional MC4R, or the complement thereof, said nucleic acid comprising a nucleic selected from the group consisting of: 
 (a) a nucleotide sequence which hybridizes under conditions of moderate stringency to the coding region of SEQ ID NO:2;    (b) a nucleotide sequence which hybridizes under conditions of moderate stringency to a polynucleotide which is complementary to the coding region of SEQ ID NO:2;    (c) a nucleotide sequence which hybridizes under conditions of moderate stringency to the coding region of the canine MC4R as deposited with the ATCC and having ATCC Accession No. PTA-1761; and    (d) a nucleotide sequence which hybridizes under conditions of moderate stringency to a polynucleotide which is complementary to the coding region of the canine MC4R as deposited with the ATCC and having ATCC Accession No. PTA-1761,    with the proviso that said functional MC4R is not human, porcine, murine, rat or chicken.    
     
     
         10 . An isolated nucleic acid encoding a functional MC4R, or the complement thereof, said nucleic acid comprising a nucleic selected from the group consisting of: 
 (a) a nucleotide sequence which hybridizes under conditions of high stringency to the coding region of SEQ ID NO:2;    (b) a nucleotide sequence which hybridizes under conditions of high stringency to a polynucleotide which is complementary to the coding region of SEQ ID NO:2;    (c) a nucleotide sequence which hybridizes under conditions of high stringency to the coding region of the canine MC4R as deposited with the ATCC and having ATCC Accession No. PTA-1761; and    (d) a nucleotide sequence which hybridizes under conditions of high stringency to a polynucleotide which is complementary to the coding region of the canine MC4R as deposited with the ATCC and having ATCC Accession No. PTA-1761.    
     
     
         11 . An isolated nucleic acid comprising a nucleotide sequence that: 
 (a) encodes a polypeptide according to SEQ ID NO:4; or    (b) encodes a polypeptide encoded by the canine MC4R clone as deposited with the ATCC and having ATCC Accession No. PTA-1761.    
     
     
         12 . The isolated nucleic acid of  claim 11 , wherein said nucleic acid has a nucleotide sequence according to SEQ ID NO:2 or the canine MC4R clone as deposited with the ATCC and having ATCC Accession No. PTA-1761.  
     
     
         13 . An isolated nucleic acid comprising a nucleotide sequence having more than 81.3% identity to SEQ ID NO:2 or the canine MC4R clone as deposited with the ATCC and having ATCC Accession No. PTA-1761.  
     
     
         14 . An isolated nucleic acid comprising a nucleotide sequence encoding a polypeptide having more than 98.1% identity to SEQ ID NO:4 or to the polypeptide encoded by the canine MC4R clone as deposited with the ATCC and having ATCC Accession No.  
     
     
         15 . An isolated nucleic acid comprising a nucleotide sequence encoding an ECD of a canine MC4R corresponding to amino acids 1-46, 98-124, 187-191, or 268-279 of SEQ ID NO:4 or of the polypeptide encoded by the canine MC4R clone as deposited with the ATCC and having ATCC Accession No. PTA-1761.  
     
     
         16 . An isolated nucleic acid comprising a nucleotide sequence encoding a CD of a canine MC4R corresponding to amino acids 69-77, 147-163, 216-244, or 302-333 of SEQ ID NO:4 or of the polypeptide encoded by the canine MC4R clone as deposited with the ATCC and having ATCC Accession No. PTA-1761.  
     
     
         17 . A nucleotide vector comprising the nucleic acid of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 ,  8 ,  9 ,  10 ,  11 ,  12 ,  13 ,  14 ,  15  or  16 .  
     
     
         18 . An expression vector comprising the nucleic acid of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 ,  8 ,  9 ,  10 ,  11 ,  12 ,  13 ,  14 ,  15  or  16  in operative association with a nucleotide regulatory element that controls expression of the polypeptide encoded by said nucleotide sequence.  
     
     
         19 . A genetically engineered host cell comprising the nucleic acid of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 ,  8 ,  9 , 10 , 11 ,  12 ,  13 ,  14 , 15  or  16 .  
     
     
         20 . A genetically engineered host cell comprising the nucleic acid of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 ,  8 ,  9 ,  10 ,  11 ,  12 ,  13 ,  14 ,  15  or  16  wherein said nucleic acid is in operative association with a nucleotide regulatory element that controls expression of said nucleotide sequence in the host cell.  
     
     
         21 . A substantially pure polypeptide encoded by the nucleic acid of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 ,  8 ,  9 , 10 , 11 ,  12 , 13 , 14 ,  15  or  16 .  
     
     
         22 . A substantially pure polypeptide comprising the amino acid sequence of: 
 (a) SEQ ID NO:3;    (b) SEQ ID NO:4;    (c) the feline MC4R clone as deposited with the ATCC and having the ATCC Accession NO. PTA-1762;    (d) the canine MC4R clone as deposited with the ATCC and having the ATCC Accession NO. PTA-1761;    (e) an ECD of a feline MC4R corresponding to amino acids 1-46, 96-123, 186-190, or 268-279 of SEQ ID NO:3 or of the polypeptide encoded by the feline MC4R clone as deposited with the ATCC and having ATCC Accession No. PTA-1762;    (f) an ECD of a canine MC4R corresponding to amino acids 1-46, 98-124, 187-191, or 268-279 of SEQ ID NO:4 or of the polypeptide encoded by the canine MC4R clone as deposited with the ATCC and having ATCC Accession No. PTA-1761;    (g) a CD of a feline MC4R corresponding to amino acids 70-76, 146-165, 212-244, or 302-333 of SEQ ID NO:3 or of the polypeptide encoded by the feline MC4R clone as deposited with the ATCC and having ATCC Accession No. PTA-1762; or    (h) a CD of a canine MC4R corresponding to amino acids 69-77, 147-163, 216-244, or 302-333 of SEQ ID NO:4 or of the polypeptide encoded by the canine MC4R clone as deposited with the ATCC and having ATCC Accession No. PTA-1761.    
     
     
         23 . An antibody that immunospecifically binds the polypeptide of  claim 21 .  
     
     
         24 . A method for producing a recombinant polypeptide, comprising: 
 (a) culturing a host cell transformed with the expression vector of  claim 18  and which expresses the recombinant polypeptide; and    (b) recovering the recombinant polypeptide from the cell culture.    
     
     
         25 . A composition comprising the polypeptide of  claim 21  and a carrier.  
     
     
         26 . A method for detecting a polynucleotide of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 ,  8 ,  9 ,  10 ,  11 , 12 , 13 , 14 , 15  or  16  in a sample, comprising: 
 (a) contacting the sample with a compound that binds to and forms a complex with the polynucleotide for a period sufficient to form the complex; and  
 (b) detecting the complex, 
 so that if a complex is detected, a polynucleotide of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 ,  8 ,  9 , 10 ,  11 , 12 , 13 ,  14 , 15  or  16  is detected.  
 
 
     
     
         27 . A method for detecting a polynucleotide of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 ,  8 ,  9 ,  10 ,  11 ,  12 ,  13 , 14 , 15  or  16  in a sample, comprising: 
 (a) contacting the sample under stringent hybridization conditions with nucleic acid primers that anneal to a polynucleotide of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 ,  8 ,  9 ,  10 ,  11 ,  12 , 13 ,  14 , 15  or  16  under such conditions; and  
 b) amplifying the annealed polynucleotides, 
 so that if a polynucleotide is amplified, the polynucleotide of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 ,  8 ,  9 ,  10 ,  11 ,  12 ,  13 ,  14 ,  15  or  16  is detected.  
 
 
     
     
         28 . The method of  claim 27 , wherein the polynucleotide is an RNA molecule that encodes a functional MC4R, and the method further comprises reverse transcribing an annealed RNA molecule into a cDNA polynucleotide.  
     
     
         29 . A method for identifying a compound that binds to the polypeptide of  claim 21 , comprising: 
 (a) contacting a compound with the polypeptide of  claim 21  for a time sufficient to form a polypeptide/compound complex; and    b) detecting the complex, 
 so that if a polypeptide/compound complex is detected, a compound that binds to a polypeptide of  claim 19  is identified.  
   
     
     
         30 . A method for identifying a compound that binds to the polypeptide of  claim 21 , comprising: 
 (a) contacting a compound with a polypeptide of  claim 21 , in a cell, for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in said cell; and    b) detecting the complex by detecting reporter gene sequence expression, 
 so that if a polypeptide/compound complex is detected, a compound that binds to a polypeptide of  claim 21  is identified.  
   
     
     
         31 . A method of modulating activity of the polypeptide of  claim 21 , comprising contacting a cell that expresses the polypeptide with a compound that modulates activity of the polypeptide for a time sufficient to modulate said activity.  
     
     
         32 . A method for screening and identifying antagonists of MC4R, comprising: 
 (a) contacting a cell line that expresses the polynucleotide of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 ,  8 ,  9 ,  10 ,  11 ,  12 ,  13 ,  14 ,  15  or  16  with a test compound in the presence of an MC4R agonist; and    (b) determining whether the test compound inhibits the binding and cellular effects of the MC4R agonist on the cell line, in which antagonists are identified as those compounds that inhibit both the binding and cellular effects of the MC4R agonist on the cell line.    
     
     
         33 . A method for screening and identifying agonists of MC4R, comprising: 
 (a) contacting a cell line that expresses the polynucleotide of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 ,  8 ,  9 ,  10 ,  11 ,  12 ,  13 ,  14 ,  15  or  16  with a test compound in the presence and in the absence of an MC4R agonist;    (b) determining whether, in the presence of the MC4R agonist, the test compound inhibits the binding of the MC4R agonist to the cell line; and    (c) determining whether, in the absence of the MC4R agonist, the test compound mimics the cellular effects of the MC4R agonist on the cell line, 
 in which agonists are identified as those test compounds that inhibit the binding but mimic the cellular effects of the MC4R agonist on the cell line.  
   
     
     
         34 . The method of  claim 33  in which the cell line is a genetically engineered cell line.  
     
     
         35 . A method for screening and identifying antagonists of MC4R comprising: 
 (a) contacting the polypeptide of  claim 21  with a random peptide library such that the polypeptide will recognize and bind to one or more peptide species within the library;    (b) isolating the polypeptide/peptide combination;    (c) determining the sequence of the peptide isolated in step (b); and    (d) determining whether the test compound inhibits the binding and cellular effects of an MC4R agonist, 
 in which antagonists are identified as those peptides that inhibit both the binding and cellular effects of the MC4R agonist.  
   
     
     
         36 . A method for screening and identifying agonists of MC4R comprising: 
 (a) contacting the polypeptide of  claim 21  with a random peptide library such that the polypeptide will recognize and bind to one or more peptide species within the library;    (b) isolating a polypeptide/peptide combination;    (c) determining the sequence of the peptide isolated in step (b); and    (d) determining whether, in the absence of a MC4R agonist, the peptide mimics the cellular effects of the MC4R agonist, 
 in which agonists are identified as those peptides that inhibit the binding of the MC4R agonist to a MC4R polypeptide but mimic the cellular effects of the MC4R agonist.  
   
     
     
         37 . A method of modulating activity of the polypeptide of  claim 21 , comprising contacting the polypeptide with a compound that modulates activity of the polypeptide for a time sufficient to modulate said activity.  
     
     
         38 . A method of modulating the endogenous enzymatic activity of MC4R in an animal comprising administering to the animal an amount of an MC4R ligand effective to modulate said endogenous enzymatic activity.  
     
     
         39 . The method of  claim 38 , wherein the animal is a cow, a pig, a goat, a sheep, a horse, a dog, or a cat.  
     
     
         40 . The method of  claim 38  in which the ligand to said MC4R receptor is an MC4R agonist.  
     
     
         41 . The method of  claim 38  in which the ligand to said MC4R receptor is an MC4R antagonist.  
     
     
         42 . The antagonist of  claim 41  that is a monoclonal antibody that immunospecifically binds to an epitope of said MC4R.  
     
     
         43 . The method of  claim 38  in which the enzymatic activity of said MC4R is increased.  
     
     
         44 . The method of  claim 38  in which the enzymatic activity of said MC4R is decreased.  
     
     
         45 . A transgenic animal in which the nucleic acid of  claim 1  or  9  is an expressed transgene contained in the genome of the animal.  
     
     
         46 . A transgenic animal in which expression of genomic sequences encoding the polypeptide of  claim 21  is prevented or repressed.  
     
     
         47 . A method for modulating the appetite and/or metabolic rate of an animal comprising administering to the animal an effective amount of an MC4R ligand.  
     
     
         48 . The method of  claim 47 , wherein the animal has an appetite-related or metabolic disorder.  
     
     
         49 . The method of  claim 48  wherein the disorder causes, is caused by, or is characterized by a reduction in appetite, feeding behavior or body weight, or an increase in metabolic rate, and the ligand is an MC4R antagonist.  
     
     
         50 . The method of  claim 49  wherein the disorder causes, is caused by, or is characterized by an increase in appetite, feeding behavior, or body weight, or a decrease in metabolic rate, and the ligand is an MC4R agonist.  
     
     
         51 . The method of  claim 49  wherein the animal is a post partum sow or dairy cow.  
     
     
         52 . The method of  claim 48  wherein the animal is a companion animal.  
     
     
         53 . The method of  claim 48  wherein the animal is a livestock animal.  
     
     
         54 . The method of  claim 48  wherein the animal is a poultry animal.  
     
     
         55 . The method of  claim 49  wherein the animal suffers from shipping or crowding stress.  
     
     
         56 . The method of  claim 49  wherein the animal is lactating.  
     
     
         57 . The method of  claim 47  wherein the animal is gravid.  
     
     
         58 . The method of  claim 49  wherein the disorder is cachexia, anorexia or weaning-induced inappetance and growth lag.  
     
     
         59 . The method of  claim 49  wherein the disorder is a metabolic disorder.  
     
     
         60 . The method of  claim 59  wherein the metabolic disorder is diabetes.  
     
     
         61 . The method of  claim 49  wherein the disorder is a disease.  
     
     
         62 . The method of  claim 61  wherein the disease is cancer, renal failure, cardiac disease, endotoxemia, fever, hepatic lipidosis, infection or inflammation.  
     
     
         63 . The method of  claim 50  wherein the animal is obese.  
     
     
         64 . The method of  claim 47  wherein the MC4R ligand is part of a pharmaceutical composition.  
     
     
         65 . A method of increasing the reproductive performance of an animal comprising administering to the animal an effective amount of an MC4R ligand.  
     
     
         66 . A method of increasing the growth performance of an animal comprising administering to the animal an effective amount of an MC4R ligand.  
     
     
         67 . The method of  claim 47  wherein the ligand is part of a pharmaceutical composition.  
     
     
         68 . The method of  claim 67  wherein the preparation is administered orally, transdermally, or by slow release subcutaneous implants/pellets.  
     
     
         69 . A kit comprising a pharmaceutical composition comprising an MC4R ligand.